The present invention is based on the surprising discovery that the diphtheria toxin fragment A can be selectively targeted to certain mammalian cell types by introduction into the cell of chimeric toxin genes in which expression of a toxin fragment A coding sequence is controlled by mammalian cell-specific regulatory sequences. The toxin fragment A coding sequence was selectively expressed in the target mammalian cell, inhibiting protein synthesis and resulting in cell death. Cell-specific expression of such chimeric toxin genes was sufficiently restricted to effect selective killing of targeted cells without elimination of non-targeted cells. It was surprising that selective lethality could be obtained using such chimeric toxin genes because there was evidence that the introduction of a single molecule of fragment A into a cell would be lethal (Yamaizumi et al. (1978) Cell 15:245-250) and it was not known, prior to the present invention, if cell-specific regulation, particularly of heterologous coding regions, would be restricted enough to cause selective lethality.
Attempts have been made to use the diphtheria toxin A fragment to selectively kill undesirable cells, such as malignant cells, without destroying healthy cells. Such attempts have concentrated on replacement of the natural fragment B protein delivery mechanism with alternate delivery mechanisms based on the specificity of certain proteins for cell surface molecules, for example by preparing toxin fragment A protein conjugates with antibodies (immunotoxins), hormones or plant lectins.
Diphtheria toxin is synthesized and secreted by strains of Corynebacterium diphtheriae which are lysogenic for bacteriophage .beta..sup.tox+. The naturally occurring toxin is a single polypeptide of about 58 kd (535 amino acids) which is highly toxic to many animal species. Diphtheria toxin inhibits protein synthesis in and is toxic to most eukaryotic cells that have been tested. The toxin is composed of two regions, separable by proteolytic cleavage, which are functionally distinct. Toxin activity is associated only with fragment A, the NH.sub.2 -terminal region of 193 amino acids. Fragment A functions by catalyzing the inactivation of elongation factor-2 (EF-2). The COOH-terminal 342 amino acid fragment B, is itself non-toxic, but functions to deliver the toxin fragment A to cells. Fragment A is non-toxic unless it is introduced into the cell cytoplasm. Purified toxin fragment A has been demonstrated to be highly toxic when introduced artificially into cells. A review of the structure and function of diphtheria toxin is provided in Pappenheimer (1977) Ann. Rev. Biochem. 46:69-94.
The diphtheria toxin (DT) gene, tox, is located on bacteriophage .beta.. The entire gene has been cloned and sequenced by separately cloning fragments having little or no toxic activity (Greenfield et al. (1983) Proc. Natl. Acad. Sci. USA 80:6853-6857). Several non-toxic mutant tox genes have also been cloned including tox45 (Leong et al. (1983) Science 220:515-517) which has a wild-type region A and non-functional B region, and tox228 (Kaczorek et al. (1983) Science 221:855-858) which carries mutations in both the A and B regions. Uchida et al. (1973) J. Biol. Chem. 248:3838-3844 and ibid. pp. 3845-3850 have identified several mutant DT proteins, designated CRM's (cross-reacting materials) which are non-toxic (CRM45, 197, 228) or have reduced toxicity (CRM176). The attenuated toxicity (about 90% of wild-type) of CRM176 results from a mutation in the A region which affects enzymatic activity of the tox176 fragment A. The coding sequence of the mature toxin is preceded by a signal sequence which presumably functions in secretion of tox gene product (Kaczorek et al. (1983)).
It is known that many differentiated eukaryotic cells synthesize proteins that are unique to a particular cell type. For example, it has been demonstrated that immunoglobulin kappa is specifically expressed in B lymphocyte cells, that interleukin-2 is selectively expressed in activated T-cells (Fujita et al. (1986) Cell 46:401-407), that gamma 2-crystallin is specifically expressed in the fiber cells of the ocular lens (i.e., Breitman et al. (1984) Proc. Natl. Acad. Sci. USA 81:7762), that elastase I is specifically expressed in pancreatic acinar cells (Ornitz et al. (1985) Nature 313:600-602), that insulin is specifically expressed in pancreatic endocrine .beta.-cells, and that chymotrypsin is specifically expressed in pancreatic exocrine cells (Walker et al. (1983) Nature 306:557-561). Additionally, there are examples of non-specific but preferential expression of transferrin (McKnight et al. (1983) Cell 34:335-341) and metallothionein in the liver. A particularly important type of cell-preferential expression occurs with certain retroviruses including human T-cell leukemia viruses, HTLV's (Sodroski et al. (1984) Science 225:381-385; Sodroski et al. ( 1985) ibid. 227:171-173) and bovine leukemia virus, BLV (Derse et al. (1985) Science 227:317-320; Rosen et al. (1985) ibid. 227:320-322). Markedly enhanced viral expression is observed in cells already infected with the virus. Selective transcription is stimulated by trans-acting regulatory factors produced in infected cells. These stimulatory factors appear to be unique for each virus. Sequences associated with control of stimulated expression have been localized to the long terminal repeat (LTR) sequence of both HTLV's and BLV. Heterologous genes placed under control of the LTR sequences are reported to be preferentially expressed in infected cells.
Chimeric genes in which a heterologous mammalian or viral structural gene is placed under the control of cell-specific regulatory elements have been reported to be successfully expressed in a cell-specific manner. An elastase-human growth hormone gene fusion was shown to be specifically expressed in pancreatic acinar cells of transgenic mice (Ornitz et al. (1985) supra). Oncogenes placed under the control of elastase and gamma A crystallin gene regulatory sequences have been shown to be specifically expressed (i.e., induce tumors) in the pancreas and ocular lens, respectively of transgenic mice (Ornitz et al. (1985) Cold Spring Harbor Symp. Quant. Biol. 50:389-409; Quaife et al. (1987) Cell 48:1023-1034; Mahon et al. (1987) Science 235:1622-1628).
A number of bacterial genes have been successfully expressed in mammalian cells under the control of mammalian promoters and regulatory sequences (see, for example, Gorman et al. (1982) Mol. Cell. Biol. 2:1044-1051; Southern and Berg (1982) J. Mol. Appl. Genet. 1:327-341). In fact, bacterial genes such as chloramphenicol acetyl transferase (CAT), aminoglycoside 3' phosphotransferase (neo), guanine phosphoribosyl transferase (gpt) and .beta.-galactosidase (lacZ) are often used as detectable or selectable markers in the study of mammalian expression systems. For example, the bacterial .beta.-galactosidase gene has been used to assess heat shock expression in Drosophila (Lis et al. (1983) Cell 35:403-410) and tissue-specific expression mediated by the gamma 2-crystallin promoter in the ocular lens of transgenic mice (Goring et al. (1987) Science 235:456-458).
Specific DNA sequences which function in cell-specific regulation have been isolated and identified in many cases. In most systems that have been studied, cell-specific expression is mediated by an enhancer, a cis-acting DNA sequence, which is believed to selectively activate expression in a target cell in response to tissue or cell-specific trans-acting factors. Immunoglobulin heavy chain (IgH) enhancers are selectively active in B-cells and are among the best characterized cell-specific expression elements (Gillies et al. (1983) Cell 33:717-728; Picard and Shaffner (1984) Nature 307:80-82; Ephrussi et al. (1985) Science 227:134-140). Enhancers are also reported to function in cell-selective expression of elastase (Hammer et al. (1987) Mol. Cell. Biol. 7:2956-2967), insulin (Edlund et al. (1985) 230:912-916) and interleukin-2 (Fujita et al. (1986)). It has recently been reported that cell-type specificity of immunoglobulin genes is conferred not only by the IgH enhancer but also by a 5'-upstream element associated with an immunoglobulin gene promoter (Mason et al. (1985) Cell 41:479-487; Foster et al. (1985) Nature 315:423-425). This upstream element apparently confers a level of cell-selective expression independent of the heavy chain enhancer. A similar 5'-upstream promoter associated element is reported to function in insulin gene regulation (Edlund et al. (1985)). In contrast, no such promoter associated element is believed to function in interleukin-2 regulation (Fujita et al. (1986)).
It has been reported (Maxwell et al. (1986) Cancer Research 46:4660-4664, which is incorporated by reference herein) that the diphtheria toxin A-chain (DT-A) gene is regulated in a cell-specific manner on transfection into human cells. This reference also reported selective killing of B-cells caused by expression of DT-A under the control of the immunoglobulin heavy chain enhancer. This reference also suggests that cell-specific regulatory mechanisms can be employed generally for selective cell killing by expression of a toxin gene and that such selective killing has application to cancer therapy. A second report (Maxwell et al. (1987) Mol. Cell. Biol. 7:1576-1579, which is incorporated by reference herein) describes the cloning and sequencing of the attenuated diphtheria toxin 176 and suggests the use of the tox176 coding region for selective cell killing.
Recently, it has been reported (Palmiter et al. (1987) Cell 50:435-443, which is incorporated by reference herein) that a chimeric diphtheria toxin fragment A coding sequence expressed under the regulatory control of an elastase I enhancer/promoter was selectively expressed in pancreatic acinar cells. Selective expression and selective lethality of the chimeric toxin gene was demonstrated by the production of transgenic mice lacking a normal pancreas. Similar results have also been obtained (Breitman et al. (1987) Science, 238:1553-1555) with diphtheria toxin fragment A under the control of gamma crystallin gene regulatory sequences, resulting in selective elimination of lens tissue in transgenic mice.
Some recently suggested approaches to therapy for Acquired Immune Deficiency Syndrome (AIDS) involve "intracellular immunization", a term coined by Baltimore ((1988) Nature 335:395-396) to describe the genetic modification of cells to render them incapable of supporting viral production. We have been exploring the use of regulated expression of a gene encoding a potent toxin, diphtheria toxin A fragment (DT-A) or an attenuated toxin fragment, to selectively kill cells infected with human immunodeficiency virus (HIV-1).
As described herein, we have placed expression of the reporter gene luciferase (luc), or of DT-A, under control of the HIV-1 trans-acting, essential Tat and Rev proteins. The Tat protein acts on a cis-acting element mapped to region +14 to +44 (referred to as the TAR region) of the HIV long terminal repeat (LTR) to increase viral expression from the LTR (Arya et al. (1985) Science 229:69-73; Rosen et al. (1985) supra; Sodroski et al. (1985) supra: Green et al. (1989) Cell 58:215-223). The Tat protein appears to exert an effect at both transcriptional (Peterlin et al. (1986) Proc. Natl. Acad. Sci. USA 83:9734-9738; Hauber et al. (1987) Proc. Natl. Acad. Sci. USA 84:6364-6368; Laspia et al. (1989) Cell 59:283-292) and post-transcriptional levels (Cullen (1986) Cell 46:973-982; Feinberg et al. (1986) Cell 46:807-817; Wright et al. (1986) Science 234:988-992; Braddock et al. (1989) Cell 58:269-279; Edery et al. (1989) Cell 56:303-312) and can stimulate expression of heterologous genes placed 3' to the TAR region (Tong-Starksen et al. (1987) Proc. Natl. Acad. Sci. USA 80:6845-6849; Felber and Pavlaskis (1988) Science 239:184-187). The Rev protein relieves the negative regulatory effect of cis-acting repressive sequences (crs) found in the env region of the HIV-1 genome (Rosen et al. (1988) Proc. Natl. Acad. Sci. USA 85:2071-2075; Hadzopoulou-Cladaras et al. (1989) J. Virol. 63:1265-1274) which repress the production of viral unspliced and singly spliced messenger RNAs (mRNAs). The Rev protein acts by binding to RNA at the Rev responsive element (RRE; Malim et al. (1989) Nature 338:254-257; Cochrane et al. (1990) Proc. Natl. Acad. Sci. USA 87:1198-1202), also localized to the env region; binding of the Rev protein to the RRE is essential for Rev function. Rev protein expression results in an increased accumulation of unspliced and singly spliced viral mRNAs, encoding structural proteins, in the cytoplasm (Felber et al. (1989) Proc. Natl. Acad. Sci. USA 86:1495-1499; Zapp and Green (1989) Cell 58:215-223). Thus, expression of the Rev protein promotes the transition from early or latent infection to productive infection. Like Tat, the Rev protein can also act in trans to activate expression of heterologous genes which contain the negative crs sequences and a correctly oriented RRE (Rosen et al. (1988) supra; Felber et al. (1989) supra). We demonstrate here that efficient regulation of both chimeric luc and chimeric DT-A expression by the Tat and Rev proteins can be achieved in transfected cells in vitro. Such regulation is applicable as a novel approach for treatment of AIDS, exploiting the extreme toxicity of DT-A to kill virus-infected cells.